The invention relates to improvements in plants. In particular, the invention relates to improved biological compositions which are effective to increase the growth rate of seedlings and develop systemic disease immunity in plants and to control soil nematodes. The invention relates also to seeds treated with the composition and to the treated seedlings and plants.
Naturally occurring nematode suppressiveness has been reported for several agricultural systems (Stirling et al., 1979, Kerry, 1982, Kluepfel et al., 1993,), but suppressiveness can also be induced by crop rotation with antagonistic plants such as switchgrass (Panicum virgatum) (Kokalis-Burelle et al., 1995) and velvetbean (Mucuna deeringiana) (Vargas et al., 1994) or organic amendments including pine bark (Kokalis-Burelle et al., 1994), hemicellulose (Culbreath et al., 1985), and chitin (Mankau and Das, 1969, Spiegel et al., 1986, Rodrxc3xadguez-Kxc3xa1bana and Morgan-Jones, 1987). A major component of the suppressiveness of chitin amendments is believed to be biotic, and several reports confirm increased numbers of nematode antagonistic microorganisms associated with chitin-induced suppressive soils (Godoy et al., 1983, Rodrxc3xadguez-Kxc3xa1bana et al., 1984). Extensive work has been done over the past years on fungi associated with chitin amendments (Godoy et al., 1983, Rodrxc3xadguez-Kxc3xa1bana et al., 1984), while less information is available on bacterial community structure and the role of bacteria in chitin-induced suppressiveness.
Chitin, a glucosamine polysaccharide, is a structural component of some fungi, insects, various crustaceans and nematode eggs. In egg shells of tylenchoid nematodes, chitin is located between the outer vitelline layer and the inner lipid layer and may occur in association with proteins (Bird and Bird, 1991). The breakdown of this polymer by chitinases can cause premature hatching which results in fewer viable juveniles (Mercer et al., 1992). In the soil, chitinases are produced by some actinomycetes (Mitchell and Alexander, 1962), fungi (Mian et al., 1982), and bacteria (Ordentlich et al., 1988, Inbar and Chet, 1991), but chitinases are also released by many plants as part of their defense mechanism against various pathogens (Punja and Zhang, 1993) and plant-parasitic nematodes (Roberts et al., 1992). Chitinases depolymerize the chitin polymer into N-acetylglucosamine and chitobiose. Further microbial activity results in the deamination of the sugar and accumulation of ammonium ions and nitrates (Rodrxc3xadguez-Kxc3xa1bana et al., 1983). Nematicidal concentrations of ammonia in association with a newly formed chitinolytic microflora are believed to cause nematode suppressiveness (Mian et al., 1982, Godoy et al., 1983). Benhamou et al. (1994) have shown that chitosan, the deacetylated derivative of chitin, induces systemic plant resistance against Fusarium oxysporum f. sp. radicis-lycopersici in tomato when applied as a seed treatment or soil amendment. This suggests that plant defense mechanisms might contribute to the overall nematode suppression.
Changes in one component of the microflora in a community often leads to other changes, and it was recently reported that soil amendment with 1% chitin led to alterations in the taxonomic structure of the bacterial communities of the soil, rhizosphere and endorhiza (Hallmann et al., 1998). Several bacterial species were found in chitin-amended soils and cotton rhizospheres which were not detected in non-amended soils and rhizospheres. Additionally, it was determined that chitin-amended soils selectively influenced the community structure of endophytic bacteria within cotton roots. For example, Phyllobacterium rubiacearum was not a common endophyte-following chitin amendment, although its populations in soil were stimulated by chitin. Burkholderia cepacia was the dominant endophyte-following chitin amendment but was rarely found among the endophytic community of non-amended plants. Hence, alterations in microbial community structure are associated with the control of nematodes which occurs upon soil amendment with chitin.
Plant-associated microorganisms have been extensively examined for their roles in natural and induced suppressiveness of soilborne diseases. Among the many groups of such organisms are root-associated bacteria, which generally represent a subset of soil bacteria. Rhizobacteria are a subset of total rhizosphere bacteria which have the capacity, upon re-introduction to seeds or vegetative plant parts (such as potato seed pieces), to colonize the developing root system in the presence of competing soil microflora. Root colonization is typically examined by quantifying bacterial populations on root surfaces; however, some rhizobacteria can also enter roots and establish at least a limited endophytic phase. Hence, root colonization may be viewed as a continuum from the rhizosphere to the rhizoplane to internal tissues of roots.
Rhizobacteria which exert a beneficial effect on the plant being colonized are termed PGPR. PGPR may benefit the host by causing plant growth promotion or biological disease control. The same strain of PGPR may cause both growth promotion and biological control. Efforts to select and apply PGPR for control of specific soilborne fungal pathogens have been reviewed (Kloepper, 1993; Glick and Bashan, 1997). Among the soilborne pathogens shown to be negatively affected by PGPR are Aphanomyces spp., Fusarium oxysporum, Gaeumannomyces graminis, Phytophthora spp., Pythium spp., Rhizoctonia solani, Sclerotium rolfsii, Thielaviopsis basicola, and Verticillium spp. In most of these cases, biological control results from bacterial production of metabolites which directly inhibit the pathogen, such as antibiotics, hydrogen cyanide, iron-chelating siderophores, and cell wall-degrading enzymes. Plant growth promotion by PGPR may also be an indirect mechanism of biological control, leading to a reduction in the probability of a plant contracting a disease when the growth promotion results in shortening the time that a plant is in a susceptible state, e.g. in the case where PGPR cause enhanced seedling emergence rate, thereby reducing the susceptible time for pre-emergence damping-off. An alternative mechanism for biological control by PGPR is induced systemic resistance.
Many recently published examples of biocontrol of nematodes by antagonists involve use of the non-culturable pathogen Pasteuria penetrans (reviewed in Stirling, 1991b). Populations of the pathogen often increase upon continual cropping of crops susceptible to nematodes and may contribute to soil suppressiveness to nematodes in these cases. P. penetrans produces resting spores which adhere to the cuticle of nematodes, where they produce a germ tube, penetrate the host, and develop an extensive colonization and digestion of the host nematode. Unfortunately, procedures to produce sufficient spores for inoculative biocontrol studies are laborious, and no practical mass cultivation systems are available (Ciancio, 1995). Nematode-trapping fungi have provided control under greenhouse conditions, however practical control in the field has not been consistently achieved. This result most likely occurs since nematode-trapping capacity of most species is not related to nematode density as would be required for economic control (Stirling, 1991a). Several reports of culturable rhizobacteria as biocontrol agents of nematodes have been published (Becker et al., 1988; Hallmann, et al., 1997; Kloepper et al., 1992; Kluepfel, et al., 1993; Martinez-Ochoa et al., 1997; Oka et al., 1993; Sikora, 1988). While some reductions in nematode damage or populations have been reported upon introduction of bacteria in these model systems, none of the studies present data showing field efficacy at levels which would provide economically practical protection.
Induced resistance, whereby a plant""s natural defenses are triggered by a physical, chemical, or biological agent, has been extensively studied and reviewed (Kuxc4x87, 1982; Ross, 1961; Ryals et al., 1996; Sticher et al. 1997; van Loon, 1997). Biological agents which induce resistance are of two general types: those that induce a necrotic lesion on the plant, indicating an incompatible pathogen-host interaction, and those that colonize the plant, usually roots, without visible necrosis. Certain rhizosphere bacteria have been shown to colonize roots of plants resulting in increased plant growth or biological control of plant diseases (reviewed in van Loon et al, 1998). Some PGPR strains, like necrosis-inducing biological agents and chemical inducers, trigger the plant""s natural defenses in response to pathogen attack. Unlike necrosis-inducing biological agents and chemical inducers which provide a short-term period of induction, PGPR colonize plant roots, thereby potentially providing an extensive period of induction. Most of these published systems use fluorescent pseudomonads, and the mechanism appears to be distinct from ISR induced by pathogens or chemicals in that pathogenisis-related (PR) proteins typically do not accumulate, and the systemic protection is most typically not dependent upon activation of salicylic acid (Press et al., 1998; reviewed in van Loon et al. 1998).
Research in support of the present invention has studied the phenomenon of PGPR-mediated ISR, and the results indicate the following points. Selected PGPR strains belonging to diverse Gram-positive and Gram-negative genera (including Pseudomonas, Serratia, and Bacillus) can, upon seed treatment or soil drench treatment to plant root systems, reduce the incidence of distally infecting pathogens. Single PGPR strains have been shown to reduce pathogen infection and symptoms of multiple diseases on cucumber and tomato. Cucumber diseases affected under both greenhouse and field studies in multiple years include foliar diseases (angular leaf spot, caused by Pseudomonas syringae pv. lachrymans [Liu et al., 1991a and b] and anthracnose [Wei et al., 1996], caused by Colletotrichurn orbiculare), systemic wilt diseases (cucurbit wilt, caused by Erwinia tracheiphila [Zehnder et al., 1997a and b] and Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum [Liu et al. 1991c]), and the systemic viral disease caused by cucumber mosaic virus (CMV) (Raupach et al., 1996; Yao et al., 1997). In the case of cucurbit wilt, disease control is linked to PGPR-mediated reductions in plant preference by the insect vectors, such as the striped and spotted cucumber beetles. In field and greenhouse studies, PGPR treatments led to significant reduction in beetle feeding (Zehnder et al., 1997a) which was associated with PGPR-mediated reductions in cucurbitacin C, a feeding attractant. With tomato, protection has been noted in the greenhouse or field against CMV, bacterial spot, caused by Xanthomonas axonopodis pv. vesicatoria; tomato mottle geminivirus, and bacterial speck, caused by P. syringae pv. tomato.
Another approach for control of nematodes which may lead to at least a limited induction of suppressiveness through microbial activity is the use of selected xe2x80x9cbotanical aromatic compoundsxe2x80x9d. xe2x80x9cBotanical aromaticsxe2x80x9d are low-molecular weight, volatile plant metabolites, many of which are found in essential oils of plants. When incorporated into soil, the volatile nature of botanical aromatics causes the compounds to act as fumigants. Furfural (2-furfuraldehyde) mixed into soil in greenhouse trials suppressed initial populations of the root-knot nematode Meloidogyne arenaria and the number of subsequent galls on squash. Similar protections were observed against M. incognita (root-knot nematode) and Heterodera glycines (cyst nematode) on soybean (Rodrxc3xadguez-Kxc3xa1bana et al., 1993). Furfural also reduced root-knot damage of okra and increased yields in a microplot trial (Rodrxc3xadguez-Kxc3xa1bana et al., 1993). Canullo et al. (1992) demonstrated that soil treatments with furfural reduced damage to the pathogenic fungus Sclerotium rolfsii on lentil, although populations of Trichoderma spp. and bacterial antagonists to S. rolfsii increased.
In a separate study with cotton, the use of the botanical aromatics furfural, benzaldehyde, and citral reduced populations of M. incognita juveniles in soil and on roots (Bauske et al., 1994). These same three botanical aromatics (furfural, citral, and benzaldehyde) have shown potential for control of both fungal pathogens and phytoparasitic nematodes (reviewed in Bauske et al., 1997). Applications of furfural and benzaldehyde to soil cause both quantitative and qualitative shifts in the composition of the soil bacterial community (reviewed Bauske et al., 1997). After decreasing in the first 24 hr after application, bacterial populations increased by 1 week after application and remained higher than in non-treated control soils for 7 weeks. There was a corresponding increase in frequency of Burkholderia cepacia in treated soils.
Soler-Serratosa et al. (1996) reported that pre-plant applications of thymol to soil reduced initial and final populations of M. incognita and H. glycines on soybean. When thymol was combined with benzaldehyde, a synergistic effect on nematode populations occurred. The effects of thymol on nematodes was related to changes in the indigenous soil microflora following treatment, and specifically to an increase in Pseudomonas spp. (Soler-Serratosa et al., 1994).
Notwithstanding this extensive work, an adequate strategy has been developed that provides satisfactory compositions suitable to treat plants from a very early stage (e.g., seeds or seedlings) to yield fast-growing plants which have a systemic resistance against foliar pathogens and alter the soil microflora to effectively control nematodes. Further alternatives to chemical pesticides and methyl bromide soil fumigation for control of plant diseases are needed. Reducing the pressure and dependence of control of plant diseases on chemical pesticide solutions is highly desired.
Therefore, there is an urgent need, both environmentally and economically, for such compositions and methods of treatment devoid of pesticides and pesticide-like elements. It is evident from the prior art that no previous disclosure nor publication has contemplated the invention in that it is possible to achieve synergistic plant growth promotion and induced systemic resistance with the present invention of PGPR and an organic amendment thereby producing plants having an increased growth rate and systemic disease immunity. This invention contributes to the solution of this need and to the problems confronting this field of art.
A bibliography of related publications appears prior to the claims section of this document.
The invention relates broadly to the field of plant growth and development, particularly to methods and compositions for enhancing plant growth and disease resistance. The invention relates to the initiation and promotion of plant growth using a combination of multiple tactics of biological control in soil or in a soil-less plant growth medium for controlling nematodes and foliar pathogens.
The invention also relates to a novel composition of a plant growth medium comprising chitin and nonchitinolytic plant growth-promoting rhizobacteria (PGPR) which creates a synergy in plant growth and disease resistance. Further the invention relates to a novel synergistic method of using either seed treatment or the application to a soil-less potting media composition of a chitinolytic element and bacteria elements for the preparation and development of plants and transplants.
The invention relates to various plant products, such as tomato and cucumber plants obtained from the invention. The invention also relates to various other compositions and plants described further below.
The invention provides several useful embodiments.
The invention provides a composition which comprises at least two PGPR bacterial strains and a chitinolytic compound or a compound of equivalent effect. The plant growth promoting rhizobacteria (PGPR) comprise at least one bacterial strain which can induce systemic plant resistance to plant diseases. The composition further comprises a chitinolytic compound which may have nematode-control activity. At least one of the PGPR bacterial strains in a preferred embodiment is non-chitinolytic. The chitinolytic compound is preferably either an aminated organic compound or an aminated polysaccharide.
Other compositions of the invention may include optional ingredients such as at least one botanical aromatic compound. Usually, a botanical aromatic compound for the present invention will have a low molecular weight and be of a volatile plant metabolite, such as citral, furfural or benzaldehyde. Numerous other compounds which do not detrimentally affect the function of the chitinolytic compound and of the PGPR may be included in the composition.
The invention provides further a method for exposing seeds and growing transplant plugs in soil or a soil-less medium, which includes the biological composition of the invention. Thereafter, the seeds and/or plugs are continued to be exposed and/or grown, respectively, in either the field or in an environment having conditions similar to that of greenhouses. These conditions, like greenhouses, may be environmentally controlled for temperature, light, humidity and the like. Alternatively, these conditions, may be free of such environmental controls. The growing continues under these conditions until a predetermined growth and size is obtained. Thereafter, the resulting plant is transferred to field conditions to continue to grow normally. The resulting plants, from either seeds or plugs, have been observed to have developed a systemic disease resistance, increased growth vigor and other desirable properties.
The method of the invention provides different means for treating a transplant seedling or seed of a target plant with the composition of the invention. One such means is to preferably spray the target seedling with an aqueous composition of the invention so as to promote the exposure and penetration of the constituents of the composition into the target plant. Another means is to expose seedlings or seeds of the target plant to the composition of the invention.
Another embodiment of the invention relates to the improved plants obtained by the invention including plants that exhibit a systemic resistance to infectious diseases, whether the infection is of the root system, the foliage or other parts of the plants.
Plants which may be treated and obtained in accordance with the invention include both monocotyledonous and dicotyledonous plant species including barley, oats, rice, wheat, soybean, corn; melons including cucumber, muskmelon, canteloupe and watermelon; vegetables including beans, pea, peanut; oil crops including canola and soybean; solanaceous plants including tobacco; tuber crops including potato; vegetables including tomato, pepper, cucumber, broccoli, cabbage, cauliflower, lettuce and radish; fruits including strawberry; fiber crops including cotton; other plants including coffee, bedding plants, perennials , woody ornamentals, turf and cut flowers including carnation and roses; sugar cane; containerized tree crops; evergreen trees including fir and pine; deciduous trees including maple and oak; and fruit trees including cherry, apple, pear and orange. In general any plant that is susceptible to plant disease and does respond to the composition of the invention may be treated in accordance with the invention.
Other embodiments of the invention will become apparent hereinafter.
The invention relates to various combinations of biocontrol tactics applied together into soil or plant growth media used for production of plants or transplant plugs. The xe2x80x9ctacticsxe2x80x9d may include mixtures of two or more PGPR strains where one or more can induce plant resistance. These xe2x80x9ctacticsxe2x80x9d may further include organic amendments, which may have nematode-control activity, and xe2x80x9cselector compoundsxe2x80x9d, such as botanical aromatic compounds (including thymol, benzaldehyde, citral, furfural, menthol and alpha-terpineol), which alter soil microflora to enhance activity of indigenous antagonistic microorganisms. The invention may also consist of using two of these three tactics, i.e. PGPR mixtures together with organic amendments, but without addition of selector compounds. The invention provides other embodiments further discussed hereinafter.
In accordance with the invention, a novel synergy of biocontrol tactics of a heretofore unidentified composition has been discovered. The composition, in accordance with the invention, initiates and promotes plant growth and synergistically induces systemic disease resistance in plants. The characteristics of the composition are both novel and synergistic. The invention provides a composition comprised of chitinolytic and nonchitinolytic constituents for plant growth and disease resistance. The composition results in a synergy of constituent characteristics including initiation and promotion of plant growth and the inducing of systemic plant disease resistance.
The composition is comprised of at least one PGPR strain and an organic amendment. The biological PGPR mixture preferably comprises spore preparations of the bacteria. Additionally, in the PGPR mixture, at least one PGPR strain can induce systemic plant resistance to disease. The presence of the bacteria in the mixture is generally on the order of 103 to 1010 bacteria per seed or per liter of soil-less mix. A preferred embodiment of the invention is where the bacteria presence is approximately 1xc3x971010 bacteria per liter of soil-less mix and 1xc3x97108 bacteria per seed.
Examples of two formulated PGPR strains include but are not limited to Bacillus sublilis strain GB03 (available from Gustafson LLC., Plano, Tex. as Kodiak(trademark)) and Bacillus amyloliquefaciens strain IN937a. Other types of nonchitinolytic bacteria suitable for the composition include: root-colonizing bacteria including the family BACILLIACEAE which is spore-forming and comprises the genera Bacillus, Paenibacillus, Brevibacillus, Virgibacillus, Alicyclobacillus and Aneurinibacillus; fluorescent pseudomonads; isolates of Pseudomonas spp., Serratia spp., Cornynebacterium spp., Enterobacterspp., Arthrobacter spp., and Burkholderia spp.; and benefical fungi such as Trichoderma spp., Gliocladium spp., and others; yeasts, and actinomycetes. Additional examples of the group of bacilli and their spore-forming genera, including Bacillus, are referenced in the xe2x80x9cATCC catalogue of Bacteriaxe2x80x9d, published by the American Type Culture Collection, and are incorporated herein by reference.
The organic amendment is also known as a chitinolytic component as it exhibits nematode-control activity. The chitinolytic component in the composition is in an amount sufficient to cause a chitinolytic effect. The amount of the chitinolytic component present may range from 0.1% to 10.0%. A preferred embodiment of the invention is where the composition is comprised of approximately 2.5% of the chitinolytic component.
Examples of the chitinolytic component include but are not limited to chitin, flaked chitin, and chitosan. The chitinolytic component can also be derived from its precursors which upon hydrolysis or other chemical or biochemical breakdown will yield the chitinolytic component. Preferably, the organic amendment is a glucose polysaccharide. Such precursors are organic natural compounds like pine bark, crab or shrimp shells, soybean meal, cotton seed meal and casein.
A further embodiment of the invention is a composition comprising at least one of the PGPR strains, an organic amendment, and an optional botanical aromatic compound. The optional addition of a botanical aromatic compound, also known as a selector compound, to the primary composition further introduces a fumigant for altering soil microflora. The presence of the fumigant reduces parasitic nematodes and increases antagonists. An example of the botanical aromatic compound includes but is not limited to benzadehyde. Other types of botanical aromatic compounds suitable for the composition include citral. Further optional components include components which do not negatively affect the function of the two principal components of the composition, referred to as non-essential ingredients.
A further embodiment of the invention is a composition comprising the two PGPR strains, an organic amendment, and an optional botanical aromatic compound.
An embodiment of the invention is a method for promoting plant growth and synergistically inducing disease resistance in plants. The method comprises exposing seed to the novel biological composition or growing seedlings in a soil-less potting media containing the novel biological composition, for a period of time sufficient to initiate improved growth and disease-suppressive characteristics. A seed is planted in the soil or in the soil-less media and grown in a greenhouse under conditions previously discussed. By way of example, the biological composition may be incorporated into a soil-less media such as in a styrofoam transplant flat and then seeded. During this growth period, if desired, additional treatments of the composition are provided to the plant in predetermined amounts at predetermined times. Then, if desired, when the seedlings have advanced to an age of approximately four to six weeks, or where the plants are ready for transplant, the plant is transplanted to field conditions or greenhouse conditions where the plant has been observed to continue to grow normally. Further, the plants are then optionally treated with the composition, in which the composition is in either a liquid or solid formn. Preferably, this treatment is in the form of a foliar spray, drench application, drip application or through irrigation where it has been observed that the plants achieve a greater resistance to disease. This optional treatment may also be performed at any time during the growth period. Further, untreated seeds are coated by exposing the seed with the composition, either in a liquid or solid form, preferably in a foliar spray, drench application, drip application or through irrigation. This treatment may be optional, and may additionally be performed at any time to untreated or treated seeds.
Particularly, this method stimulates and promotes plant growth in the early seedling stages which is known to be a difficult stage of plant growth to stimulate with PGPR alone. The method also induces disease resistance systemically in plants to a greater extent than previously achieved in the prior art. The invention also synergistically enables both the stimulation and promotion of plant growth in combination with systemically protecting a plant from disease by exposure to the biological composition.
Further embodiments of the invention provide methods for inhibiting the growth of disease agent Phytophtora infestans (a causal agent of late blight disease), Xanthomonas axonopodis pv. vesicatoria (a causal agent of bacteria spot disease), Pseudomonas syringae pv. lachrymans (a causal agent of angular leaf spot disease), and Fusarium spp. by using the method discussed above and challenging the plant with a treatment of the disease agent. The challenged seedling is then evaluated and the incidence and severity of disease are measured . It has been observed that plants grown under the present invention and challenged with the disease exhibited a significantly greater ability to resist the disease.
An additional embodiment of the invention is a plant which having been exposed to the composition exhibits the synergistic effects of improved growth in combination with the systemic protection from disease. Seeds, seedling and plants which may be treated and obtained in accordance with the invention include both monocotyledonous and dicotyledonous plant species including barley, oats, rice, wheat, soybean, corn; melons including cucumber, muskmelon, cantaloupe and watermelon; vegetables including beans, pea, peanut; oil crops including canola and soybean; solanaceous plants including tobacco; tuber crops including potato; vegetables including tomato, pepper, cucumber, broccoli, cabbage, cauliflower, lettuce and radish; fruits including strawberry; fiber crops including cotton; other plants including coffee, bedding plants, perennials , woody ornamentals, turf and cut flowers including carnation and roses; sugar cane; containerized tree crops; evergreen trees including fir and pine; deciduous trees including maple and oak; and fruit trees including cherry, apple, pear and orange. In general any seed, seedling or plant that is susceptible to plant disease and does respond to the composition of the invention may be treated in accordance with the invention.
The resulting plant of the present invention exhibits improved plant growth in the early seedling stages, which is known to be a difficult stage of plant growth to stimulate with PGPR alone. The plant further exhibits measurably improved plant physical characteristics such as greater height, weight, vigor, leaflets and leaflet surface area, at growth stages earlier than non-treated, control plants. Similarly, the plant displays less disease than untreated, control plants.
Further embodiments of the invention include various media for culturing the plants using the composition, in particular tomato, cucumber, and classes previously discussed, and parts thereof which have been developed in accordance with the invention.
Other embodiments of the invention will become apparent in the further detailed description of preferred and other embodiments of the invention.
The term xe2x80x9ctransplantxe2x80x9d as used herein is a term of art used to designate a plant of any age of any variety that is moved from one growing location to another.
The term xe2x80x9ctransplant plugxe2x80x9d as used herein is a plant of any age situated in a contained growth medium in which the plant is prepared to be transplanted or transported from one location to another.
The term xe2x80x9csoil-less mediumxe2x80x9d as used herein is a growth medium comprising peat-based products which may contain perlite, vermiculite, a fertilizer component and other ingredients. Examples of a soil-less medium include readily available products such as xe2x80x9cPro-mixxe2x80x9d, xe2x80x9cRedi-Groxe2x80x9d and xe2x80x9cSpeedling Mixxe2x80x9d.
The term xe2x80x9cseedlingxe2x80x9d as used herein is a term of art used to designate a plant of an age ranging from the day of emergence to one year after planting or an age where transplant of the plant occurs and is not limited to or by any plant class. The term also includes xe2x80x9cforest seedlingsxe2x80x9d which can be one year old.
The term xe2x80x9csynergyxe2x80x9d as used herein is used to designate the resulting action of two or more substances to achieve an effect of which each is individually incapable of achieving.